It is generally implied that phenotypic properties of transformed and malignant cells are genetically controlled. Cytoplasmic factors, however, may be able to regulate some of these phenotypic properties. We propose to study the role of the cytoplasm in the expression of parameters of transformation, including contact inhibition, serum dependency, plating efficiency in solid substrate, cell saturation and, particularly, the loss of anchorage dependence. Simultaneously, the role of the cytoplasm in "in vivo" tumorigenicity in nude mice will be examined. These objectives will be achieved by fusing transformed enucleated 501-1 mouse fibroblasts carrying the cytoplasmic genetic marker for chloramphenicol resistance (cytoplasts), with nontransformed K22 rat liver epithelial cells or with their "nuclei" (karyoplasts). Conversely, nontransformed cytoplasts obtained from 1007-AR1 rat liver epithelial cells, also chloramphenicol resistant, will be fused with intact transformed mouse L929 fibroblasts or with their karyoplasts. The products of fusion (termed "cybrids" and "nucybrids," respectively) will be assayed for the above parameters of transformation and inoculated in nude mice. The existence of cytoplasmic factors transferable through fusion will be evaluated by the acquisition or repression of the specified parameters of transformation, and by the generation of tumors in nude mice. The persistence of these changes will also be explored, by similar methods, at various times after the isolation of cybrid and nucybrid clones. Several laboratories have recently reported on viable proliferating clones developed from both cybrids and nucybrids. Thus, technological aspects of this proposal should be facilitated considerably. Identification of putative cytoplasmic factors is not a goal of this specific project, but it is contemplated for a future application based on data obtained in this one.